lets, the metabolism of the liberated arachidonic acid by cycloox- ygenase activity to ... lipoxygenase activity to 12-hydroxy-5,8,10,14-icosatetraenoic acid.
Proc. Nati Acad. Sci. USA Vol. 79, pp. 7709-7713, December 1982 Biochemistry
Cytochalasins inhibit arachidonic acid metabolism in thrombinstimulated platelets (actin polymerization/phospholipases C and A2/cyclooxygenase/lipoxygenase/phosphatidic acid)
WOLFGANG SIESS*, EDUARDO G. LAPETINAt, AND PEDRO CUATRECASAS Department of Molecular Biology, The Wellcome Research Laboratories, 3030 Cornwallis Road, Research Triangle Park, North Carolina 27709
Contributed by Pedro Cuatrecasas, September 27, 1982
and thromboxanes in activated platelets has been implicated in platelet aggregation and serotonin release. In this work we studied the possible correlation between changes in lipids and cytoskeleton in thrombin-stimulated platelets. Cytochalasins reduce actin polymerization and, also, the metabolism of arachidonic acid in thrombin-activated platelets. MATERIALS AND METHODS [1-'4C]Arachidonic acid (58 mCi/mmol; 1 Ci = 3.7 X 10'° becquerels) and L-a-phosphatidyl[U-'4C]inositol (270 mCi/ mmol) were from Amersham and [1,2-3H]serotonin (26.4 Ci/ mmol) was from New England Nuclear. Cytochalasin B and D were obtained from Sigma and kept in dimethyl sulfoxide (Me2SO) at -20°C. Thrombin was purchased from Sigma. Other materials were obtained as documented (20). Preparation of Washed Horse Platelets Labeled with [14C]Arachidonic Acid and [3H]Serotonin. Platelet-rich plasma was obtained from 500 ml of horse blood treated to prevent coagulation with 71 mM citric acid/85 mM trisodium citrate/Ill mM dextrose as described (20, 21). Platelet-rich plasma (280 ml) was then double-labeled with 15 ,uCi of [14C]arachidonic acid and 150 ,uCi of [3H]serotonin by incubating for 2 hr at 37°C. Platelets were separated from plasma by centrifugation at 3,000 x g for 20 min at 4°C and washed twice with ice-cold buffer (1mM EGTA/20 mM Hepes/134 mM NaCl/3.5 mg of albumin per ml, pH 7.4) as described (20). The platelets were finally resuspended in 25 ml of buffer, corresponding to a platelet concentration of 1.5-2.0 X 109 per ml. Incubations were carried out in a shaking incubator water bath at 37°C. Platelet suspensions were incubated before addition of thrombin (termed "preincubated") for 5 min at 37°C with Me2SO or cytochalasin B. The final concentration of Me2SO was 0.2%, and it did not affect the thrombin-induced release of serotonin or that of arachidonic acid plus its metabolites. Then, thrombin (0.15 unit or 0.5 unit/ml) was added for 5 min. For measuring the production of ['4C]arachidonic acid plus its metabolites and the release of [3H]serotonin, incubations of platelets (0.2 ml) were performed in Eppendorf microtubes and stopped by centrifugation in a Beckman Microfuge B for 1 min. The 14C or 3H radioactivity in the supernatant was measured by liquid scintillation counting in a two-channel counter. The cross-interference of the two channels was negligible (3% for 14C; 0.03% for 3H). For measurement of the different [14C]arachidonic acid metabolites, samples (0. 8 ml) were extracted with 3.0 ml ofchloroform/ methanol, 1:2 (vol/vol), and the phases were partitioned with
ABSTRACT Low concentrations (0.5-1 puM) of cytochalasins inhibit the thrombin-stimulated polymerization of monomeric actin to filamentous actin in platelets. Similar concentrations of cytochalasin B inhibit the formation and metabolism of arachidonic acid in horse platelets stimulated by low concentrations of thrombin (0.1-0.5 unit/ml). However, the release of serotonin is not inhibited by cytochalasin B. Cytochalasins B and D (0.5-1 JAM) markedly reduce, in thrombin-stimulated human or horse platelets, the metabolism of the liberated arachidonic acid by cyclooxygenase activity to thromboxane B2 and 12-hydroxy-5,8,10-heptadecatrienoic acid and the conversion of arachidonic acid by lipoxygenase activity to 12-hydroxy-5,8,10,14-icosatetraenoic acid. The generation of arachidonic acid from platelet phospholipids and the formation of phosphatidic acid are much less affected by cytochalasin B or D. Cytochalasins do not directly inhibit platelet cyclooxygenase, lipoxygenase, phospholipase A2, or phosphatidylinositol-specific phospholipase C. In addition, the metabolism of exogenously added arachidonic acid by intact platelets is not inhibited by cytochalasins B and D. The results indicate that polymerization of actin in platelets stimulated by thrombin may be required for the effective metabolism of arachidonic acid released from platelet phospholipids.
Polymerization of monomeric actin to filamentous actin occurs rapidly after stimulation of platelets with thrombin (1-3). Actin polymerization may be related to the early morphological change of platelets from discoid, smooth-shaped cells to spiny spheres with protrusion of pseudopods (4, 5), which precedes platelet responses such as spreading, adhesion, and aggregation. Cytochalasins are fungal metabolites that inhibit actin polymerization by binding to the growing end of actin filaments and blocking the further addition of monomeric actin molecules (69). It has been shown (5, 10) that cytochalasins inhibit actin polymerization or induce depolymerization of formed actin filaments in thrombin-stimulated platelets. Cytochalasins are reported to inhibit platelet shape change and, in higher concentrations, could also prevent platelet adhesion and aggregation (11-13). However, the results concerning the effects of cytochalasins on the release of serotonin from platelets are controversial (14, 15). Another early biochemical response to platelet stimulation is the activation of phospholipases C and A2. Stimulation of phospholipase C leads to the formation of 1,2-diacylglycerol and phosphatidic acid, both ofwhich may play a role as intracellular mediators (16-18). Stimulation of phospholipases A2 leads to the liberation of arachidonic acid from membrane phospholipids and the immediate degradation of arachidonic acid by cyclooxygenase and lipoxygenase (19). The formation of endoperoxides
Abbreviations: Me2SO, dimethyl sulfoxide; PGI2, prostacyclin; TXB2, thromboxane B2; 12-HETE, 12-hydroxy-5,8, 10,14-icosatrienoic acid; 12-HHT, 12-hydroxy-5,8, 10,-heptadecatrienoic acid. * Present address: Medizinische Klinik Innenstadt Der Universitat Munchen Munich, Federal Republic of Germany. t To whom reprint requests and correspondence should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 7709
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1.0 ml of chloroform and 1.0 ml of water. The lower phase was dried under nitrogen, and arachidonic acid metabolites were separated by thin-layer chromatography as described (21). Specific areas were localized by autoradiography, and `4C radioactivity was measured by liquid scintillation counting. Preparation of Washed Human Platelets Labeled with [14C]Arachidonic Acid. Platelets were prepared from fresh human blood treated to prevent coagulation with 0.15 vol of the citric acid/citrate/dextrose buffer. Blood donors had not received any medication in the previous 4 wk. Platelet-rich plasma (pH 6.4) was obtained by centrifugation at 200 x g for 20 min. Prostacyclin (PGI2) was used for the separation ofplatelets from plasma to prevent activation of human platelets (22). Isolation ofplatelets was performed at room temperature. Platelet-rich plasmawas labeled with [ 4C]arachidonic acid (0.25 ,uCi/ ml) for 25 min at 37TC. Then, PGI2 (5 ng/ml) was added, and the platelets were centrifuged at 800 x g for 15 min. Platelets were resuspended and washed twice in buffer (134 mM NaCl/ 12 mM NaHCO3/2.9 mM KCl/0.36 mM NaH2PO4/5 mM Hepes/5 mM glucose/i mM EDTA, pH 7.4) containing PGI2 (300 ng/ml) by centrifugation at 600 X g for 10 min. Platelets were finally resuspended in buffer without PGI2, the concentration being adjusted to 7.5 x 108 platelets per ml. Platelets were used 20 min after final resuspension. Samples (0.5 ml) were preincubated with Me2SO (final concentration, 0.2%) or cytochalasin D for 5 min at 37°C in a shaking incubator water bath and were stimulated with thrombin (0.2 unit/ml). Incubations were stopped, and lipids were extracted by adding 3.75 vol of 1:2 chloroform/methanol. The phases were partitioned by adding 1.25 vol of chloroform and 1.25 vol of 0.1% formic acid. ['4C]Arachidonic acid metabolites from the lower phases were separated by thin-layer chromatography (21), localized by autoradiography with EN3HANCE spray (New England Nuclear) and intensifying screen, and measured by liquid scintillation counting. Metabolism of Exogenous [I4C]Arachidonic Acid by Platelets. Horse or human washed platelets (0.5 ml) were incubated with different concentrations of cytochalasin B or D for 5 min before exposure to [I4C]arachidonic acid (5 /LM) for 2 min. Samples were extracted and products were separated by thin-layer chromatography as described above. Measurement of Enzyme Activities. Cyclooxygenase and lipoxygenase activities were measured in lysed horse platelets incubated with ['4C]arachidonic acid (10 ,tM) for 2 min, and reaction products were separated by thin-layer chromatography (23). Phospholipase C activity was assayed in the supernatant separated from homogenized horse platelets by centrifugation at 100,000 X g. Assays contained 20 ,ug of cytosolic protein, 0.5 mM L-phosphatidyl['4C]inositol, and 1 mM Ca2O in 0.1 ml of buffer (25 mM Tris maleate/100 mM NaCl, pH 5.5). After 5 min, reactions were stopped with 0.37 ml of 1:2 chloroform/ methanol, and phases were partitioned by the addition of 0.12 ml of chloroform and 0.12 ml of water. [14C]Inositol phosphates produced during the reaction were measured in the upper, aqueous phase by liquid scintillation counting. Phospholipase A2 was assayed in the particulate fraction separated at 100,000 x g from homogenized horse platelets. Assays contained 75 ,ug of membrane protein, 0.5 mM L-a-phosphatidyl[14C]inositol, and 1 mM Ca2' in 0.1 ml of 0.1 M Tris (pH 9.0). After 10 min, reactions were stopped with 0.37 ml of chloroform/methanol/ concentrated HC1, 100:200:2 (vol/vol), followed by addition of 0.12 ml of chloroform and 0.12 ml of 2 M KC1 and separation of the phases. Lipids from the lower phases were dried under N2 and phosphatidylinositol was separated from lysophosphatidylinositol on thin-layer chromatography (24).
Proc. Natl. Acad. Sci. USA 79 (1982) 100 r
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Cytochalasin B, M FIG. 1. Cytochalasin B inhibits the generation of arachidonic acid and its metabolites (e, A) into the medium of platelets stimulated with thrombin, whereas the release of serotonin (o, A) is unaffected. Horse platelets were double-labeled with ["4Clarachidonic acid and [3H]serotonin, preincubated with 0.2% Me2SO (control) or 0.2% cytochalasin B in 0.2% Me2SO for 2 min, and stimulated for 5 min with 0.2 unit (A, A) or 0.5 unit (e, o) of thrombin per ml. ['4C]Arachidonic acid plus its metabolites and [3H]serotonin were measured in the supernatant after centrifugation of platelets. The released [3H]serotonin in control platelets stimulated with 0.2 and 0.5 unit of thrombin per ml constituted 6.2% and 32% of the total present, respectively. The equivalent values for ['4C]arachidonic acid and its metabolites were 6% and 12%, respectively.
NaDodSO4/Polyacrylamide Electrophoresis of Platelet Cytoskeleton. Washed human platelets were preincubated with cytochalasin D or Me2SO for 5 min and stimulated with thrombin (0.2 unit/ml) for 1 min. Platelets were then lysed by adding an equal volume of Triton X-100 extraction buffer (2% Triton 100
-
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FIG. 2. Cytochalasin B inhibits the thrombin-induced metabolism of arachidonic acid in horse platelets. Platelets labeled with ["4C]arachidonic acid were preincubated with Me2SO (control) or cytochalasin B in Me2SO and then were stimulated with 0.15 unit (a) or 0.5 unit (o) of thrombin per ml for 5 min. AA, arachidonic acid; PA, phosphatidic acid. Values for [14C]arachidonic acid plus ['4C]arachidonic acid metabolites in controls (without cytochalasin) were 4% and 13% of the total platelet-labeled arachidonate after stimulation with 0.15 and 0.5 unit of thrombin per ml, respectively.
Biochemistry:
Proc. Natl. Acad. Sci. USA 79 (1982)
Siess et aL
7711
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FIG. 4. NaDodSO4/polyacrylamide gel electrophoresis of Triton X-100-insoluble platelet cytoskeleton from control (lanes b-d) and thrombin-activated (lanes e-j) platelets pretreated with (lanes f-j) and without (lane e) cytochalasin D. Lanes: a, platelet homogenate; b, nonincubated platelets; c, platelets incubated with 0.2% Me2SO; d, platelets incubated with cytochalasin D (10 kLM); e, platelets preincubated in 0.2% Me2SO and stimulated with thrombin (0.2 unit/ml) for 1 min; f-i, platelets preincubated for 5 min with increasing concentrations of cytochalasin D in 0.2% Me2SO (0.1, 1.0, 10.0, 100 ,uM, respectively) before stimulation with thrombin; j, platelets preincubated with 10 tM cytochalasin D for 10 sec before stimulation with thrombin. ABP, actinbinding protein.
2
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FIG. 3. Inhibition of thrombin-stimulated arachidonate metabolism by cytochalasin D in human platelets. Platelets labeled with ["4C]arachidonic acid were preincubated with 0.2% Me2SO (.) or 5 .M cytochalasin D in 0.2% Me2SO (o) and then were stimulated with thrombin (0.2 unit/ml). TXB2, thromboxane B2; DG, 1,2-diacylglycerol; AA, arachidonic acid; PA, phosphatidic acid. Production of ["C]arachidonic acid plus arachidonic acid metabolites with thrombin stimulation without cytochalasin D was 1.8% of total platelet arachidonic acid after 6- and 10-min incubations.
X-100/137 mM NaCl/10 mM Tris/10 mM EGTA, pH 7.0) and, after 5 min, were centrifuged in a Beckman Microfuge B for 4 min (2). The Triton X-100-insoluble pellet was washed twice with buffer, solubilized in Tris buffer containing 2% NaDodSO4 (wt/vol) and 2% 2-mercaptoethanol (wt/vol) and incubated at 100°C for 10 min. Aliquots containing 10-100 ,jg of protein were electrophoresed through slab gels (25), and proteins were stained with Coomassie brilliant blue. RESULTS Low concentrations (0.5-1 uM) of cytochalasin B inhibited the liberation or metabolism, or both, of arachidonic acid in horse platelets stimulated by thrombin (Fig. 1). In contrast, the release of serotonin was not affected by these concentrations of cytochalasin B. The inhibitory action of cytochalasin B was considerably more evident on the metabolism of arachidonic acid by cyclooxygenase and lipoxygenase than on the liberation of
arachidonic acid from platelet phospholipids or the formation of phosphatidic acid; the formation of the cyclooxygenase product, 12-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT), and the lipoxygenase product, 12-hydroxy-5,8,10,14-icosatetraenoic acid (12-HETE), was affected more drastically than was the formation of phosphatidic acid and the liberation of arachidonic acid (Fig. 2). The effect of cytochalasin B was more pronounced at low concentrations of thrombin (0.15 unit/ml compared to 0.5 unit/ml). When the thrombin concentration was raised to 1 unit/ml, which resulted in maximal activation of platelets, even much higher concentrations (100 p.M) of cytochalasin B did not affect the metabolism of arachidonic acid (data not shown). Cytochalasin D produced effects similar to those of cytochalasin B (Fig. 3). Again, the formation of cyclooxygenase and lipoxygenase products was very strongly inhibited, whereas Table 1. Effect of cytochalasin B on enzymes involved in the liberation or metabolism of arachidonic acid in horse platelets Enzyme activity, Metabolite formation, nmol/min per nmol/min/109 platelets mg protein 12Phospho- Phospho- 12Additions lipase A2 lipase C HHT TXB2 HETE 1.05 6.0 41 0.65 0.41 None 1.15 0.43 0.65 6.9 43 Me2SO, 0.5% Cytochalasin B 44 0.60 0.41 1.1 0.5 ,M 6.8 1.15 7.3 43 0.65 0.43 5.0 gM 44 0.42 1.1 50.0 ,LM 6.9 0.62 1.0 0.39 45 0.63 250.0 ,uM 6.9 0.70 0.40 1.1 6.4 48 500.0 ,uM Phospholipase A2 activity was measured in the total particulate fraction, and phospholipase C activity, in the soluble fraction obtained from horse platelet homogenates. The metabolism of exogenous [14C]_ arachidonic acid in platelet homogenates was determined by the formation of metabolites derived from cyclooxygenase (12-HHT and TXB2) and from lipoxygenase (12-HETE) activities.
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Table 2. Metabolism of exogenous ['4C]arachidonic acid by intact human or horse platelets preincubated with cytochalasins B and D Metabolite formation, nmol/min/109 platelets 12-HETE 12-HHT Additions TXB2 Horse platelets 0.47 0.35 0.61 None 0.38 0.45 0.61 Me2SO, 0.2% Cytochalasin B, 0.48 0.39 0.62 0.5 ,uM 0.44 0.36 0.59 5.0 AuM 0.30 0.49 0.6 50.0 ,uM 0.41 0.35 0.64 250.0 /AM 0.43 0.41 500.0 ,uIM 0.61 Human platelets 1.3 1.30 2.5 None 1.5 2.4 1.20 Me2SO, 0.2% Cytochalasin D, 1.4 1.12 2.3 0.04 XAM 1.15 1.3 2.4 0.2 ,uM 1.4 1.12 2.4 0.4 pM 1.2 1.12 2.3 2.0 uM 1.2 1.12 2.3 4.0 /uM 1.2 2.2 1.00 20.0 IpM 1.2 1.00 2.3 40.0 p&M The metabolism of exogenous ["4C]arachidonic acid by intact human or horse platelets was measured by the formation of cyclooxygenase (12-HHT, TXB2) and lipoxygenase (12-HETE) metabolites. Platelets were preincubated with the indicated concentrations of cytochalasin B or D and then exposed to 5 pM [(4Clarachidonic acid for 5 min.
the formation of phosphatidic acid and 1,2-diacylglycerol plus arachidonic acid were only minimally affected. The thrombin-induced polymerization of actin in human platelets also was inhibited by cytochalasin D (Fig. 4). The effective concentration range of cytochalasin D which caused the reduction of filamentous actin in the Triton X-100-insoluble platelet cytoskeleton was 1-10 puM (Fig. 4). Cytochalasin B or D did not inhibit directly the platelet enzyme activities that are involved in the liberation of metabolism of arachidonic acid (Tables 1 and 2). The cyclooxygenase, lipoxygenase, phospholipase A2, and phospholipase C activities of horse platelets were unaffected by a broad range of concentrations of cytochalasin B (Table 1). The direct metabolism of exogenously added arachidonic acid by intact horse or human platelets also was unaffected by cytochalasin B or D (Table 2). DISCUSSION The present studies show that low concentrations of cytochalasin B or D inhibit the metabolism of endogenously produced arachidonic acid in horse or human platelets stimulated by thrombin. This action suggests a blockade of arachidonic acid metabolism because the liberation of arachidonic acid from platelet phospholipids and the formation of [14C]arachidonylphosphatidic acid are inhibited to a much lesser extent in horse or human platelets. The effective concentrations of cytochalasin B or D (1 or 5 ,u M, respectively) are in the same range as those required for the inhibition of actin polymerization in platelets (refs. 5 and 10; Fig. 4). In contrast, cytochalasin does not affect the thrombin-induced release of serotonin (Fig. 1), indicating a dissociation between actin polymerization and serotonin release. The data suggest that the mechanism by which cytochalasins interfere with the metabolism of arachidonic acid in thrombin-
Proc. Natl. Acad. Sci. USA 79 (1982)
stimulated platelets may be related to the inhibition of actin polymerization by cytochalasins. Notably, cytochalasin B does not directly affect any ofthe platelet enzyme activities involved in the liberation and metabolism of arachidonic acid, such as phospholipase A2, phospholipase C, cyclooxygenase, and lipoxygenase (Table 1), and exogenously added arachidonic acid is metabolized without interference by intact platelets in the presence of cytochalasins (Table 2). Thus, the action of cytochalasin on the metabolism of arachidonic acid is clearly indirect. Furthermore, the inhibition is only observed if the extent of platelet activation is low (0.15-0.5 unit/ml of thrombin in the presence of EGTA or EDTA and with no stirring), and it can be overcome with high thrombin concentrations (1 unit/ml), even in the presence of high concentrations of cytochalasin. Cytochalasins are known to bind preferentially to actin filaments, and they are believed to prevent further actin polymerization by capping the fast-growing end of the actin filament (6-9). Recent observations indicate, however, that the action of cytochalasins on actin polymer formation in vitro and cytoskeletal networks in vivo is more complex than believed previously (26, 27). Resting platelets contain only a few short microfilaments (28-30), whereas activated platelets contain an organized cytoskeletal structure with nets or bundles of microfilaments (2, 27, 28, 30). In activated platelets, cytochalasins might act by preventing the formation of long filaments or by disrupting the network organization with formation of actin filament foci (27). Thrombin stimulates the liberation from membrane phospholipids of arachidonic acid, which is then rapidly metabolized by cyclooxygenase and lipoxygenase activities (31, 32). The effects of cytoskeletal disruption on the thrombin-induced formation of cyclooxygenase and lipoxygenase metabolites must occur by interference with the systems by which the liberated arachidonic acid reaches the specific intracellular sites of its metabolism. The fact that free arachidonic acid is not increased could suggest some inhibition of phospholipases of the A2 type or stimulation of reesterification of free arachidonic acid into platelet phospholipids. Alternatively, this also could reflect disruption of a normal positive feedback mechanism between the metabolism and production of arachidonic acid. Notably, the absence of any effects of the cytochalasins on the metabolism of exogenously added arachidonic acid compared to the endogenously produced arachidonic acid stresses dramatically the differences in the metabolic pathways that must exist. Thus, there is strong reason to suspect that endogenously produced arachidonic acid is delivered (or closely coupled) specifically to sites of this metabolism and is not released as "free" arachidonic acid. The present studies suggest that polymerization of actin in platelets may be closely linked with the metabolism of arachidonic acid in activated platelets. 1. Carlsson, L., Markey, F., Blikstad, I., Persson, T. & Lindberg, U. (1979) Proc. Natl Acad. Sci. USA 76, 6376-6380. 2. Jennings, L. K., Fox, J. E. B., Edwards, H. H. & Phillips, D. R. (1981)J. BiOd Cheim. 256, 6927-6932.
3. Pribluda, V., Lamb, F. & Rotman, A. (1981) Eur. J. Biochem. 116, 293-296. 4. Boyle Kay, M. M. & Fudenberg, H. H. (1973) Nature (London)
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5. Casella, J. F., Flanagan, M. D. & Lin, S. (1981) Nature (London) 293, 302-305. 6. Brown, S. S. & Spudich, J. A. (1979)J. Cell Bio. 83, 657-662. 7. MacLean-Fletcher, S. & Pollard, T. D. (1980) Cel 20, 329-341. 8. Brenner, S. L. & Korn, E. D. (1979) J. Biol Chem. 254, 99829985. 9. Flanagan, M D. & Lin, S. (1980)J. BioL Chem. 255, 835-838. 10. Fox, J. E. B. & Phillips, D. R. (1981) Nature (London) 292, 650652.
Biochemistry: Siess et al. 11. Peerschke, E. I. & Zucker, M. B. (1980) Thromb. Haemostasis Gen. Inf. 43, 58-60. 12. Cazenave, J. P., Packham, M. A., Guccione, M. A. & Mustard, J. F. (1974) J. Lab. Clin. Med. 84, 483-493. 13. Harfenist, E. J., Packham, M. A., Kinlough-Rathbone, R. L. & Mustard, J. F. (1981)J. Lab. Clin. Med. 97, 680-688. 14. Haslam, R. J., Davidson, M. M. L. & McClenaghan, M. D. (1975) Nature (London) 253, 455-457. 15. Kirkpatrick, J. P., McIntire, L. V., Moake, J. L. & Cimo, P. L. (1979) Thromb. Haemostasis Gen. Inf. 42, 1483-1489. 16. Rittenhouse-Simmons, S. (1979) J. Clin. Invest. 63, 580-587. 17. Lapetina, E. G., Billah, M. M. & Cuatrecasas, P. (1981) Nature (London) 292, 367-369. 18. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. & Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276. 19. Bills, T. K., Smith, J. B. & Silver, M. J. (1976) Biochim. Biophys. Acta 424, 303-314. 20. Billah, M. M., Lapetina, E. G. & Cuatrecasas, P. (1980)J. Biol. Chem. 255, 10227-10231.
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21. Lapetina, E. G. & Cuatrecasas, P. (1979) Biochim. Biophys. Acta 573, 394-401. 22. Vargas, J. R., Radomski, M. & Moncada, S. (1982) Prostaglandins 23, 929-945. 23. Siegel, M. I., McConnell, R. T. & Cuatrecasas, P. (1979) Proc. NatL. Acad. Sci. USA 76, 3774-3778. 24. Billah, M. M. & Lapetina, E. G. (1982)J. Biol. Chem. 257, 51965200. 25. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 26. Tellam, R. & Frieden, C. (1982) Biochemistry 21, 3207-3214. 27. Schliwa, M. (1982) J. Cell Biol 92, 79-91. 28. Nachmias, V. T. (1980) J. Cell Biol 86, 795-802. 29. Gonnella, P. A. & Nachmias, V. T. (1981)J. Cell Biol. 89, 146151. 30. White, J. G. (1971) in Platelet Aggregation, ed. Caen, J. (Masson, Paris), pp. 15-52. 31. Lapetina, E. G. (1982) Trends Pharmacol. Sci. 3, 115-118. 32. Lapetina, E. G., Billah, M. M. & Cuatrecasas, P. (1981) J. Biol. Chem. 256, 5037-5040.
